GSM6729390: Young, Input, replicate2; Mus musculus; RIP-Seq
Total RNA extraction was performed from each individual hippocampal tissue with NucleoZol and NucleoSpin RNA Set (Macherey-Nagel) with rDNase treatment. Three biological replicates per age group were selected from the pre-validation qPCR assay and subjected to further poly(A)-RNA enrichment using Oligo d(T)25 magnetic beads (NEB). Approximately 1.4 μg Poly(A)-RNA were chemically fragmented to ~100 nt using NEBNext RNA fragmentation buffer (NEB). RNA immunoprecipitation with the m6A-specific antibody (cat no. 202 003; Synaptic System) was performed as previously described (Widagdo et al., 2016). The input and immunoprecipitated RNA were converted to libraries using the SMARTer Stranded Total RNA-Seq Kit v2 Pico Input Mammalian (Takara Bio) and their dual combinatorial indexes for multiplexing.